RCA-free adenoviral vector system and propagation method

ABSTRACT

The present invention provides multiply deficient adenoviral vectors and complementing cell lines. Also provided are recombinants of the multiply deficient adenoviral vectors and a therapeutic method, particularly relating to gene therapy, vaccination, and the like, involving the use of such recombinants.

“This is a continuation of copending U.S. patent application Ser. No.08/757,023, filed on Nov. 26, 1996, which is a continuation of U.S.patent application Ser. No. 08/258,416, filed Jun. 10, 1994.”

TECHNICAL FIELD OF THE INVENTION

The present invention relates to recombinant, multiply deficientadenoviral vectors and complementing cell lines and to the therapeuticuse of such vectors.

BACKGROUND OF THE INVENTION

During the winter and spring of 1952-1953, Rowe and his colleagues atthe National Institutes of Health (NIH) obtained and placed in tissueculture adenoids that had been surgically removed from young children inthe Washington, D.C. area (Rowe et al., Proc. Soc. Exp. Biol. Med., 84,570-573 (1953)). After periods of several weeks, many of the culturesbegan to show progressive degeneration characterized by destruction ofepithelial cells. This cytopathic effect could be serially transmittedby filtered culture fluids to established tissue cultures of human celllines. The cytopathic agent was called the “adenoid degenerating” (Ad)agent. The name “adenovirus” eventually became common for these agents.The discovery of many prototype strains of adenovirus, some of whichcaused respiratory illnesses, followed these initial discoveries (Roweet al., supra; Dingle et al., Am. Rev. Respir. Dis., 97, 1-65 (1968);reviewed in Horwitz, “Adenoviridae and their replication,” In Virology,Fields et al., eds., 2nd ed., Raven Press Ltd., New York, N.Y., pp.1679-1721 (1990)).

Over 40 adenoviral subtypes have been isolated from humans and over 50additional subtypes have been isolated from other mammals and birds(reviewed in Ishibashi et al., “Adenoviruses of animals,” In TheAdenoviruses, Ginsberg, ed., Plenum Press, New York, N.Y., pp. 497-562(1984); Strauss, “Adenovirus infections in humans,” In The Adenoviruses,Ginsberg, ed., Plenum Press, New York, N.Y., pp. 451-596 (1984)). Allthese subtypes belong to the family Adenoviridae, which is currentlydivided into two genera, namely Mastadenovirus and Aviadenovirus. Alladenoviruses are morphologically and structurally similar. In humans,however, adenoviruses show diverging immunological properties and are,therefore, divided into serotypes. Two human serotypes of adenovirus,namely Ad2 and Ad5, have been studied intensively and have provided themajority of information about adenoviruses in general.

Adenoviruses are nonenveloped, regular icosahedrons, 65-80 nm indiameter, consisting of an external capsid and an internal core. Thecapsid is composed of 20 triangular surfaces or facets and 12 vertices(Horne et al., J. Mol. Biol., 1, 84-86 (1959)). The facets are comprisedof hexons and the vertices are comprised of pentons. A fiber projectsfrom each of the vertices. In addition to the hexons, pentons, andfibers, there are eight minor structural polypeptides, the exactpositions of the majority of which are unclear. One minor polypeptidecomponent, namely polypeptide IX, binds at positions where it canstabilize hexon-hexon contacts at what is referred to as thegroup-of-nine center of each facet (Furcinitti et al., EMBO, 8,3563-3570 (1989)). The minor polypeptides VI and VIII are believed tostabilize hexon-hexon contacts between adjacent facets, and the minorpolypeptide IIIA, which is known to be located in the regions of thevertices, is suggested to link the capsid and the core (Stewart et al.,Cell, 67, 145-154 (1991)).

The viral core contains a linear, double-stranded DNA molecule withinverted terminal repeats (ITRs), which vary in length from 103 bp to163 bp (Garon et al., PNAS USA 69, 2391-2394 (1972); Wolfson et al.,PNAS USA, 69, 3054-3057 (1972); Arrand et al., J. Mol. Biol., 128,577-594 (1973); Steenberg et al., Nucleic Acids Res., 4, 4371-4389(1977); and Tooze, DNA Tumor Viruses, 2nd ed., Cold Spring Harbor, N.Y.:Cold Spring Harbor Laboratory. pp. 943-1054 (1981)). The ITRs harbororigins of DNA replication (Garon et al., supra; Wolfson et al., supra;Arrand et al., supra; Steenberg et al., supra). The viral DNA isassociated with four polypeptides, namely V, VII, μ, and terminalpolypeptide (TP). The 55 kd TP is covalently linked to the 5′ ends ofthe DNA via a dCMP (Rekosh et al., Cell, 11, 283-295 (1977); Robinson etal., Virology, 56, 54-69 (1973)). The other three polypeptides arenoncovalently bound to the DNA and fold it in such a way as to fit itinto the small volume of the capsid. The DNA appears to be packaged intoa structure similar to cellular nucleosomes as seen from nucleasedigestion patterns (Corden et al., PNAS USA, 73, 401-404 (1976); Tate etal., Nucleic Acids Res., 6, 2769-2785 (1979); Mirza et al., Biochim.Biophys. Acta, 696, 76-86 (1982)).

The overall organization of the adenoviral genome is conserved amongserotypes, such that specific functions are similarly positioned. TheAd2 and Ad5 genomes have been completely sequenced and sequences ofselected regions of genomes from other serotypes are available.

Adenovirus begins to infect a cell by attachment of the fiber to aspecific receptor on the cell membrane (Londberg-Holm et al., J. Virol.,4, 323-338 (1969); Morgan et al., J. Virol., 4, 777-796 (1969); Pastanet al., “Adenovirus entry into cells: some new observations on an oldproblem,” In Concerts in Viral Pathogenesis, Notkins et al., eds.,Springer-Verlag, New York, N.Y., pp. 141-146 (1987)). Then, the pentonbase binds to an adenoviral integrin receptor. The receptor-bound virusthen migrates from the plasma membrane to clathrin-coated pits that formendocytic vesicles or receptosomes, where the pH drops to 5.5 (Pastan etal., Concepts in Viral Pathogenesis, Notkins and Oldstone, eds.Springer-Verlag, N.Y. pp. 141-146 (1987)). The drop in pH is believed toalter the surface configuration of the virus, resulting in receptosomerupture and release of virus into the cytoplasm of the cell. The viralDNA is partially uncoated, i.e., partially freed of associated proteins,in the cytoplasm while being transported to the nucleus.

When the virus reaches the nuclear pores, the viral DNA enters thenucleus, leaving most of the remaining protein behind in the cytoplasm(Philipson et al., J. Virol., 2, 1064-1075 (1968)). However, the viralDNA is not completely protein-free—at least a portion of the viral DNAis associated with at least four viral polypeptides, namely V, VII, TPand μ, and is converted into a viral DNA-cell histone complex (Tate etal., Nucleic Acids Res., 6, 2769-2785 (1979)).

The cycle from cell infection to production of viral particles lasts 1-2days and results in the production of up to 10,000 infectious particlesper cell (Green et al., Virology, 13, 169-176 (1961)). The infectionprocess of adenovirus is divided into early (E) and late (L) phases,which are separated by viral DNA replication, although some events whichtake place during the early phase also take place during the late phaseand vice versa. Further subdivisions have been made to fully describethe temporal expression of viral genes.

During the early phase, viral mRNA, which constitutes a minor proportionof the total RNA present in the cell, is synthesized from both strandsof the adenoviral DNA present in the cell nucleus. At least fiveregions, designated E1-4 and MLP-L1, are transcribed (Lewis et al.,Cell, 7, 141-151 (1976); Sharp et al., Virology, 75, 442-456 (1976);Sharp, “Adenovirus transcription,” In The Adenoviruses, Ginsberg, ed.,Plenum Press, New York, N.Y., pp. 173-204 (1984)). Each region has adistinct promoter(s) and is processed to generate multiple mRNA species,and, therefore, each region may be thought of as a gene family.

The products of the early (E) regions serve regulatory roles for theexpression of other viral components, are involved in the generalshut-off of cellular DNA replication and protein synthesis, and arerequired for viral DNA replication. The intricate series of eventsregulating early mRNA transcription begins with expression of immediateearly regions E1A, L1 and the 13.5 kd gene (reviewed in Sharp (1984),supra; Horwitz (1990), supra). Expression of the delayed early regionsE1B, E2A, E2B, E3 and E4 is dependent on the E1A gene products. Threepromoters, the E2 promoter at 72 map units (mu), the protein IXpromoter, and the IVa promoter are enhanced by the onset of DNAreplication but are not dependent on it (Wilson et al., Virology, 94,175-184 (1979)). Their expression characterizes an intermediate phase ofviral gene expression. The result of the cascade of early geneexpression is the start of viral DNA replication.

Adenoviral DNA replication displaces one parental single-strand bycontinuous synthesis in the 5′ to 3′ direction from replication originsat either end of the genome (reviewed in Kelly et al., “Initiation ofviral DNA replication,” In Advances in Virus Research, Maramorosch etal., eds., Academic Press, Inc., San Diego, Calif., 34: 1-42 (1988);Horwitz (1990), supra; van der Vliet, “Adenovirus DNA replication invitro,” In The Eukarvotic Nucleus, Strauss et al., eds., Telford Press,Caldwell, N.J. 1: 1-29 (1990)). Three viral proteins encoded from E2 areessential for adenoviral DNA synthesis: the single-stranded DNA bindingprotein (DBP), the adenoviral DNA polymerase (Ad pol), and thepre-terminal protein (pTP). In addition to these, in vitro experimentshave identified many host cell factors necessary for DNA synthesis.

DNA synthesis is initiated by the covalent attachment of a dCMP moleculeto a serine residue of pTP. The pTP-dCMP complex then functions as theprimer for Ad pol to elongate. The displaced parental single-strand canform a panhandle structure by base-pairing of the inverted terminalrepeats. This terminal duplex structure is identical to the ends of theparental genome and can serve as an origin for the initiation ofcomplementary strand synthesis.

Initiation of viral DNA replication appears to be essential for entryinto the late phase. The late phase of viral infection is characterizedby the production of large amounts of the viral structural polypeptidesand the nonstructural proteins involved in capsid assembly. The majorlate promoter (MLP) becomes fully active and produces transcripts thatoriginate at 16.5 mu and terminate near the end of the genome.Post-transcriptional processing of this long transcript gives rise tofive families of late mRNA, designated L1-5 (Shaw et al., Cell, 22,905-916 (1980)). The mechanisms which control the shift from the earlyto late phase and result in such a dramatic shift in transcriptionalutilization are unclear. The requirement for DNA replication may be acis-property of the DNA template, since late transcription does notoccur from a superinfecting virus at a time when late transcription ofthe primary infecting virus is active (Thomas et al., Cell, 22, 523-533(1980)).

Assembly of the virion is an intricate process from the first step ofassembling major structural units from individual polypeptide chains(reviewed in Philipson, “Adenovirus Assembly,” In The Adenoviruses,Ginsberg, ed., Plenum Press, New York, N.Y. (1984), pp. 309-337; Horwitz(1990), supra). Hexon, penton base, and fiber assemble into trimerichomopolymer forms after synthesis in the cytoplasm. The 100 kd proteinappears to function as a scaffolding protein for hexon trimerization andthe resulting hexon trimer is called a hexon capsomer. The hexoncapsomeres can self-assemble to form the shell of an empty capsid, andthe penton base and fiber trimers can combine to form the penton whenthe components are inside the nucleus. The facet of the icosahedron ismade up of three hexon capsomeres, which can be seen by dissociation ofthe capsid, but the intermediate step of formation of a group-of-ninehexons has not been observed. Several assembly intermediates have beenshown from experiments with temperature-sensitive mutants. Theprogression of capsid assembly appears dependent on scaffoldingproteins, 50 kd and 30 kd, and the naked DNA most probably enters thenear-completed capsid through an opening at one of the vertices. Thelast step of the process involves the proteolytic trimming of theprecursor polypeptides pVI, pVII, pVIII and pTP, which stabilizes thecapsid structure, renders the DNA insensitive to nuclease treatment, andyields a mature virion.

Recombinant adenoviral vectors have been used in gene therapy. The useof a recombinant adenoviral vector to transfer one or more recombinantgenes enables targeted delivery of the gene or genes to an organ,tissue, or cells in need of treatment, thereby overcoming the deliveryproblem encountered in most forms of somatic gene therapy. Furthermore,recombinant adenoviral vectors do not require host cell proliferationfor expression of adenoviral proteins (Horwitz et al., In Virology,Raven Press, New York, 2, 1679-1721 (1990); and Berkner, BioTechniques,6, 616 (1988)) and, if the diseased organ in need of treatment is thelung, has the added advantage of being normally trophic for therespiratory epithelium (Straus, In Adenoviruses, Plenum Press, New York,pp. 451-496 (1984)).

Other advantages of adenoviruses as potential vectors for human genetherapy are as follows: (i) recombination is rare; (ii) there are noknown associations of human malignancies with adenoviral infectionsdespite common human infection with adenoviruses; (iii) the adenoviralgenome (which is linear, double-stranded DNA) currently can bemanipulated to accommodate foreign genes ranging in size from smallpeptides up to 7.0-7.5 kb in length; (iv) an adenoviral vector does notinsert its DNA into the chromosome of a cell, so its effect isimpermanent and unlikely to interfere with the cell's normal function;(v) the adenovirus can infect non-dividing or terminally differentiatedcells, such as cells in the brain and lungs; and (vi) live adenovirus,having as an essential characteristic the ability to replicate, has beensafely used as a human vaccine (Horwitz, M. S. et al.; Berkner et al.;Straus et al.; Chanock et al., JAMA, 195, 151 (1966); Haj-Ahmad et al.,J. Virol., 57, 267 (1986); and Ballay et al., EMBO, 4, 3861 (1985)).

Until now, adenoviral vectors used to express a foreign gene have beendeficient in only a single early region (E1) that is essential forviral'growth, i.e., singly functionally deficient. Only the essentialregion E1 or, alternatively, the nonessential region E3 has been removedfor insertion of a foreign gene into the adenoviral genome. If theregion removed from the adenovirus is essential for the virus to grow, acomplementing system, such as a complementing cell line is necessary tocompensate for the missing viral function. In other words, thecomplementing cell line will express the missing viral function so thatthe singly deficient adenovirus can grow inside the complementing cell.Currently, there are only a few cell lines that exist that willcomplement for essential functions missing from a singly deficientadenovirus. Examples of such cell lines include HEK-293 (Graham et al.,Cold Sprina Harbor Symp. Quant. Biol., 39, 637-650 (1975)), W162(Weinberg et al., PNAS USA, 80, 5383-5386 (1983)), and gMDBP (Klessig etal., Mol. Cell. Biol., 4, 1354-1362 (1984); Brough et al., Virology,190, 624-634 (1992)).

Foreign genes have been inserted into two major regions of theadenoviral genome for use as expression vectors. Insertion into the E1region results in defective progeny that require either growth incomplementary cells or the presence of an intact helper virus (Berkneret al., J. Virol., 61, 1213-1220 (1987); Davidson et al., J. Virol., 61,1226-1239 (1987); and Mansour et al., Mol. Cell Biol., 6, 2684-2694(1986)). This region of the genome has been used most frequently forexpression of foreign genes. Such E1-defective expression vector virusesusually have been grown in the HEK-293 cell line, which contains andexpresses the complementing adenoviral E1 region. The inserted geneshave been placed under the control of various promoters and most producelarge amounts of the foreign gene product, dependent on the expressioncassette. These adenoviral vectors, however, are defective innoncomplementing cell lines. In contrast, the E3 region is nonessentialfor virus growth in tissue culture, and replacement of this region witha foreign gene expression cassette leads to a virus that canproductively grow in a noncomplementing cell line. The insertion andexpression of the hepatitis B surface antigen in the E3 region withsubsequent inoculation and formation of antibodies in the hamster hasbeen reported (Morin et al., PNAS USA, 84, 4626-4630 (1987)).

The problem with singly deficient adenoviral vectors is that they limitthe amount of usable space within the adenoviral genome for insertionand expression of a foreign gene. Due to similarities, or overlap, inthe viral sequences contained within the singly deficient adenoviralvectors and the complementing cell lines that currently exist,recombination events can take place and create replication competentviruses within a vector stock. This event can render a stock of vectorunusable for gene therapy purposes as a practical matter.

Accordingly, it is an object of the present invention to providemultiply deficient adenoviral vectors that can accommodate insertion andexpression of larger pieces of foreign DNA. It is another object of thepresent invention to provide cell lines that complement the presentinventive multiply deficient adenoviral vectors. It is also an object ofthe present invention to provide recombinants of multiply deficientadenoviral vectors and therapeutic methods, particularly relating togene therapy, vaccination, and the like, involving the use of suchrecombinants. These and other objects and advantages of the presentinvention, as well as additional inventive features, will be apparentfrom the following detailed description.

BRIEF SUMMARY OF THE INVENTION

The present invention provides multiply deficient adenoviral vectors andcomplementing cell lines. The multiply deficient adenoviral vectors canaccommodate insertion and expression of larger fragments of foreign DNAthan is possible with currently available singly deficient adenoviralvectors. The multiply deficient adenoviral vectors are also replicationdeficient, which is particularly desirable for gene therapy and othertherapeutic purposes. Accordingly, the present invention also providesrecombinant multiply deficient adenoviral vectors and therapeuticmethods, for example, relating to gene therapy, vaccination, and thelike, involving the use of such recombinants.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a set of schematic diagrams of the Ad_(GV)CFTR.10L andAd_(GV)CFTR.10R viral vectors.

FIG. 2 is a set of schematic diagrams of the Ad_(GV)CFTR.11 viralvectors.

FIG. 3 is a schematic diagram of the Ad_(GV)CFTR.12 viral vector.

FIG. 4 is a schematic diagram of the Ad_(GVC)FTR.13 viral vector.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides, among other things, multiply deficientadenoviral vectors for gene cloning and expression. The multiplydeficient adenoviral vectors of the present invention differ fromcurrently available singly deficient adenoviral vectors in beingdeficient in at least two essential gene functions and in being able toaccept and express larger pieces of foreign DNA.

Any subtype, mixture of subtypes, or chimeric adenovirus may be used asthe source of DNA for generation of the multiply deficient adenoviralvectors. However, given that the Ad5 genome has been completelysequenced, the present invention will be described with respect to theAd5 serotype.

Preferably, the adenoviral vector of the present invention is at leastdeficient in a function provided by early region 1 (E1), which includesearly region 1A (E1A) and early region 1B (E1B), and/or one or morefunctions encoded by early region 2 (E2), such as early region 2A (E2A)and early region 2B (E2B), and/or early region 3 (E3), and/or earlyregion 4 (E4) of the adenoviral genome. Any one of the deletedfunctional regions then may be replaced with a promoter-variableexpression cassette to produce a novel gene product. The insertion of anovel gene into the E2A region, for example, may be facilitated by theintroduction of a unique restriction site, such that the novel geneproduct may be expressed from the E2A promoter.

The present invention, however, is not limited to adenoviral vectorsthat are deficient in gene functions only in the early region of thegenome. Also included are adenoviral vectors that are deficient in thelate region of the genome, adenoviral vectors that are deficient in theearly and late regions of the genome, as well as vectors in whichessentially the entire genome has been removed, in which case it ispreferred that at least either the viral inverted terminal repeats andsome of the promoters or the viral inverted terminal repeats and apackaging signal are left intact. One of ordinary skill in the art willappreciate that the larger the region of the adenoviral genome that isremoved, the larger the piece of exogenous DNA that can be inserted intothe genome. For example, given that the adenoviral genome is 36 kb, byleaving the viral inverted terminal repeats and some of the promotersintact, the theoretical capacity of the adenovirus is approximately 35kb. Alternatively, one could generate a multiply deficient adenoviralvector that contains only the ITR and a packaging signal. This couldthen effectively allow for expression of 37-38 kb of foreign DNA fromthis vector.

In general, virus vector construction relies on the high level ofrecombination between fragments of adenoviral DNA in the cell. Two orthree fragments of adenoviral DNA, containing regions of similarity (oroverlap) between fragments and constituting the entire length of thegenome, are transfected into a cell. The host cell's recombinationmachinery constructs a full-length length viral vector genome. Similarprocedures for constructing viruses containing alterations in varioussingle regions have been previously described (Berkner et al., NucleicAcids Res., 12, 925-941 (1984); Berkner et al., Nucleic Acids Res., 11,6003-6020 (1983); Brough et al., Virol., 190, 624-634 (1992)) and can beused to construct multiply deficient viruses as can in vitrorecombination and ligation, for example.

The first step in virus vector construction is to construct a deletionor modification of a particular region of the adenoviral genome in aplasmid cassette using standard molecular biological techniques. Afterextensive analysis, this altered DNA (containing the deletion ormodification) is then moved into a much larger plasmid that contains upto one half of the adenovirus genome. The next step is to transfect theplasmid DNA (containing the deletion or modification) and a large pieceof the adenovirus genome into a recipient cell. Together these twopieces of DNA encompass all of the adenovirus genome plus a region ofsimilarity. Within this region of similarity a recombination event willtake place to generate a complete intact viral genome with the deletionor modification. In the case of multiply deficient vectors, therecipient cell will provide not only the recombination functions butalso all missing viral functions not contained within the transfectedviral genome. The multiply deficient vector can be further modified byalteration of the ITR and/or packaging signal, for example, such thatthe multiply deficient vector only functions in a complementing cellline.

In addition, the present invention also provides complementing celllines for propagation of the present inventive multiply deficientadenoviral vectors. The preferred cell lines of the present inventionare characterized in complementing for at least one gene function of thegene functions comprising the E1, E2, E3 and E4 regions of theadenoviral genome. Other cell lines include those that complementadenoviral vectors that are deficient in at least one gene function fromthe gene functions comprising the late regions, those that complementfor a combination of early and late gene functions, and those thatcomplement for all adenoviral functions. One of ordinary skill in theart will appreciate that the cell line of choice would be one thatspecifically complements for those functions that are missing from therecombinant multiply deficient adenoviral vector of interest and thatcan be generated using standard molecular biological techniques. Thecell lines are further characterized in containing the complementinggenes in a nonoverlapping fashion, which eliminates the possibility ofthe vector genome recombining with the cellular DNA. Accordingly,replication-competent adenoviruses are eliminated from the vectorstocks, which are, therefore, suitable for certain therapeutic purposes,especially gene therapy purposes. This also eliminates the replicationof the adenoviruses in noncomplementing cells.

The complementing cell line must be one that is capable of expressingthe products of the two or more deficient adenoviral gene functions atthe appropriate level for those products in order to generate a hightiter stock of recombinant adenoviral vector. For example, it isnecessary to express the E2A product, DBP, at stoichiometric levels,i.e., relatively high levels, for adenoviral DNA replication, but theE2B product, Ad pol, is necessary at only catalytic levels, i.e.,relatively low levels, for adenoviral DNA replication. Not only must thelevel of the product be appropriate, the temporal expression of theproduct must be consistent with that seen in normal viral infection of acell to assure a high titer stock of recombinant adenoviral vector. Forexample, the components necessary for viral DNA replication must beexpressed before those necessary for virion assembly. In order to avoidcellular toxicity, which often accompanies high levels of expression ofthe viral products, and to regulate the temporal expression of theproducts, inducible promoter systems are used. For example, the sheepmetallothionine inducible promoter system can be used to express thecomplete E4 region, the open reading frame 6 of the E4 region, and theE2A region. Other examples of suitable inducible promoter systemsinclude, but are not limited to, the bacterial lac operon, thetetracycline operon, the T7 polymerase system, and combinations andchimeric constructs of eukaryotic and prokaryotic transcription factors,repressors and other components. Where the viral product to be expressedis highly toxic, it is desirable to use a bipartite inducible system,wherein the inducer is carried in a viral vector and the inducibleproduct is carried within the chromatin of the complementing cell line.Repressible/inducible expression systems, such as the tetracyclineexpression system and lac expression system also may be used.

DNA that enters a small proportion of transfected cells can becomestably maintained in an even smaller fraction. Isolation of a cell linethat expresses one or more transfected genes is achieved by introductioninto the same cell of a second gene (marker gene) that, for example,confers resistance to an antibiotic, drug or other compound. Thisselection is based on the fact that, in the presence of the antibiotic,drug, or other compound, the cell without the transferred gene will die,while the cell containing the transferred gene will survive. Thesurviving cells are then clonally isolated and expanded as individualcell lines. Within these cell lines are those that will express both themarker gene and the genes of interest. Propagation of the cells isdependent on the parental cell line and the method of selection.Transfection of the cell is also dependent on cell type. The most commontechniques used for transfection are calcium phosphate precipitation,liposome, or DEAE dextran mediated DNA transfer.

Many modifications and variations of the present illustrative DNAsequences and plasmids are possible. For example, the degeneracy of thegenetic code allows for the substitution of nucleotides throughoutpolypeptide coding regions, as well as in the translational stop signal,without alteration of the encoded polypeptide coding sequence. Suchsubstitutable sequences can be deduced from the known amino acid or DNAsequence of a given gene and can be constructed by conventionalsynthetic or site-specific mutagenesis procedures. Synthetic DNA methodscan be carried out in substantial accordance with the procedures ofItakura et al., Science, 198, 1056 (1977) and Crea et al., PNAS USA, 75,5765 (1978). Site-specific mutagenesis procedures are described inManiatis et al., Molecular Cloning: A Laboratorv Manual, Cold SpringHarbor, N.Y. (2d ed. 1989). Therefore, the present invention is in noway limited to the DNA sequences and plasmids specifically exemplified.Exemplified vectors are for gene therapy of cystic fibrosis and,therefore, contain and express the CFTR gene but the vectors describedare easily convertible to treat other potential diseases including, butnot limited to, other chronic lung diseases, such as emphysema, asthma,adult respiratory distress syndrome, and chronic bronchitis, as well ascancer, coronary heart disease, etc. Accordingly, any gene or DNAsequence can be inserted into a multiply deficient adenoviral vector.The choice of gene or DNA sequence should be one that will achieve atherapeutic effect, for example, in the context of gene therapy,vaccination, and the like.

One skilled in the art will appreciate that suitable methods ofadministering a multiply deficient adenoviral vector of the presentinvention to an animal for therapeutic purposes, e.g., gene therapy,vaccination, and the like (see, for example, Rosenfeld et al., Science,252, 431-434 (1991), Jaffe et al., Clin. Res., 39(2), 302A (1991),Rosenfeld et al., Clin. Res., 39(2), 311A (1991), Berkner,BioTechniques, 6, 616-629 (1988)), are available, and, although morethan one route can be used to administer the vector, a particular routecan provide a more immediate and more effective reaction than anotherroute. Pharmaceutically acceptable excipients are also well-known tothose who are skilled in the art, and are readily available. The choiceof excipient will be determined in part by the particular method used toadminister the composition. Accordingly, there is a wide variety ofsuitable formulations of the pharmaceutical composition of the presentinvention. The following formulations and methods are merely exemplaryand are in no way limiting. However, oral, injectable and aerosolformulations are preferred.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the compound dissolved indiluents, such as water, saline, or orange juice; (b) capsules, sachetsor tablets, each containing a predetermined amount of the activeingredient, as solids or granules; (c) suspensions in an appropriateliquid; and (d) suitable emulsions. Tablet forms can include one or moreof lactose, mannitol, corn starch, potato starch, microcrystallinecellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellosesodium, talc, magnesium stearate, stearic acid, and other excipients,colorants, diluents, buffering agents, moistening agents, preservatives,flavoring agents, and pharmacologically compatible excipients. Lozengeforms can comprise the active ingredient in a flavor, usually sucroseand acacia or tragacanth, as well as pastilles comprising the activeingredient in an inert base, such as gelatin and glycerin, or sucroseand acacia, emulsions, gels, and the like containing, in addition to theactive ingredient, such excipients as are known in the art.

The vectors of the present invention, alone or in combination with othersuitable components, can be made into aerosol formulations to beadministered via inhalation. These aerosol formulations can be placedinto pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like. They also maybe formulated as pharmaceuticals for non-pressured preparations such asin a nebulizer or an atomizer.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid excipient, for example, water, for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions can be prepared from sterile powders, granules, and tabletsof the kind previously described.

Additionally, the vectors employed in the present invention may be madeinto suppositories by mixing with a variety of bases such as emulsifyingbases or water-soluble bases.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams, or spray formulascontaining, in addition to the active ingredient, such carriers as areknown in the art to be appropriate.

The dose administered to an animal, particularly a human, in the contextof the present invention will vary with the gene or other sequence ofinterest, the composition employed, the method of administration, andthe particular site and organism being treated. The dose should besufficient to effect a desirable response, e.g., therapeutic or immuneresponse, within a desirable time frame.

The multiply deficient adenoviral vectors and complementing cell linesof the present invention also have utility in vitro. For example, theycan be used to study adenoviral gene function and assembly.

The following examples further illustrate the present invention and, ofcourse, should not be construed as in any way limiting its scope.Enzymes referred to in the examples are available, unless otherwiseindicated, from Bethesda Research Laboratories (BRL), Gaithersburg, Md.20877, New England Biolabs Inc. (NEB), Beverly, Mass. 01915, orBoehringer Mannheim Biochemicals (BMB), 7941 Castleway Drive,Indianapolis, Ind. 46250, and are used in substantial accordance withthe manufacturer's recommendations. Many of the techniques employedherein are well known to those in the art. Molecular biology techniquesare described in detail in laboratory manuals, such as Maniatis et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y. (2d ed.1989) and Current Protocols in Molecular Biology (Ausubel et al., eds.(1987)). One of ordinary skill in the art will recognize that alternateprocedures can be substituted for various procedures presented below.Although the examples and figures relate to Ad_(GV).10, Ad_(GV).11,Ad_(GV).12, and Ad_(GV).13 which contain the cystic fibrosistransmembrane regulator gene (CFTR), namely Ad_(GV)CFTR.10,Ad_(GV)CFTR.11, Ad_(GV)CFTR.12, and Ad_(GV)CFTR.13, these vectors arenot limited to expression of the CFTR gene and can be used to expressother genes and DNA sequences. For example, therefore, the presentinvention encompasses such vectors comprising any foreign gene (e.g.,for use in gene therapy), any DNA sequence capable of expressing in amammal a polypeptide capable of eliciting an immune response to thepolypeptide (e.g., for use in vaccination), and any DNA sequence capableof expressing in a mammal any other therapeutic agent (e.g., anantisense molecule, particularly an antisense molecule selected from thegroup consisting of mRNA and a synthetic oligonucleotide).

EXAMPLE 1

This example describes the generation of one embodiment involvingAD_(GV).10, namely Ad_(GV)CFTR.10.

Ad_(GV)CFTR.10 expresses the CFTR gene from the cytomegalovirus (CMV)early promoter. Two generations of this vector have been constructed andare designated Ad_(GV)CFTR.10L and Ad_(GV)CFTR.10R, dependent on thedirection in which the CFTR expression cassette is placed in the E1region in relation to the vector genome as shown in FIG. 1, which is aset of schematic diagrams of Ad_(GV)CFTR.10L and Ad_(GV)CFTR.10R.

The CFTR expression cassette was constructed as follows. pRK5 (GenentechInc., South San Francisco, Calif.) was digested with Kpn I (New EnglandBiolabs (NEB), Beverly, Mass.), blunt-ended with Mung Bean nuclease(NEB), and an Xho I linker (NEB) was ligated in place of the Kpn I site.The resulting vector was named pRK5-Xho I. pRK5-Xho I was then digestedwith Sma I (NEB) and Hin dIII (NEB) and blunt-ended with Mung beannuclease. A plasmid containing the CFTR gene, pBQ4.7 (Dr. Lap-Chee Tsui,Hospital for Sick Children, Toronto, Canada), was digested with Ava I(NEB) and Sac I (NEB) and blunt-ended with Mung bean nuclease. These twofragments were isolated and ligated together to produce pRK5-CFTR1, theCFTR expression cassette.

pRK5-CFTR1 was digested with Spe I (NEB) and Xho I and blunt-ended withKlenow (NEB). pAd60.454 (Dr. L. E. Babiss, The Rockefeller University,New York, N.Y.), which contains Ad5 sequences from 1-454/3325-5788, wasdigested with Bql II (NEB) and blunt-ended with Klenow. These twofragments were purified from vector sequences by low-melt agarosetechnique (Maniatis et al., Molecular Cloning: a laboratory manual, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y., 2nd ed. (1989)) andligated together to produce the left arm plasmids PGVCFTR.10L andpGVCFTR.10R.

The left arm plasmid from pGVCFTR.10L or pGVCFTR.10R was digested withNhe I (NEB). The right arm of the virus was produced by digestingAd5d324 (Dr. Thomas E. Shenk, Princeton University, Princeton, N.J.)with Cla I (NEB). The two fragments, a small 918 bp fragment and a largeapproximately 32,800 bp fragment, were separated by sucrose gradientcentrifugation (Maniatis et al., supra). The large fragment was mixedwith the left arm plasmid fragments and transfected into 293 cells bystandard calcium phosphate protocol (Graham et al., Virology, 52, 456(1973)). The resulting recombinant viruses were plaque-purified on 293cells, and viral stocks were established using standard virologytechniques (e.g., Berkner et al., (1983) and (1984), supra).

EXAMPLE 2

This example describes the generation of Ad_(Gv)CFTR.11.

Ad_(Gv)CFTR.11 was constructed by means of a single in vivorecombination between 1-27082, i.e., the left arm, of Ad_(GV)CFTR.10 anda plasmid (pGV11A, pGV11B, pGV11C, or pGV11D; described in detail below)containing 21562-35935, i.e., the right arm, of Ad5 linearized with BamHI (NEB) and Sal I (NEB) and into which the various E3 and E4alterations as described below were introduced.

The left arm from Ad_(GV)CFTR.10 was isolated on a concave 10-40%sucrose gradient, wherein ¼th of the total solution was 40%, afterintact Ad_(GV)CFTR.10 was digested with Spe I (NEB) and Srf I(Stratagene, La Jolla, Calif.) to yield the 1-27082 bp fragment.

The right arm was obtained by Bam HI-Sal I digestion of a modified pGEMvector (pGBS). pGBS was generated as follows. pGemI (Promega, Madison,Wis.) was digested with Eco RI and blunt-ended with Klenow, and a Sal Ilinker was ligated into the vector. The resulting DNA was then digestedwith Sal I and religated, thereby replacing the Eco RI site with a Sal Isite and deleting the sequence between the two Sal I sites, to generatepGEMH/P/S, which was digested with Hin dIII and blunt-ended with Klenow,and a Bam HI linker was ligated into the vector to generate pGEMS/B.pGEMS/B was digested with Bam HI and Sal I and ligated with an ˜14 kbBam HI-Sal I fragment (21562-35935 from Ad5) from a pBR plasmid calledp50-100 (Dr. Paul Freimuth, Columbia University, NY) to generate pGBS.

Three different versions of the right arm plasmid have been constructedin order to introduce into the adenoviral vector two Ad E3 gene productshaving anti-immunity and anti-inflammatory properties. The large E3deletion in pGBSΔE3ORF6, designated pGV11(O) (Example 7), wasessentially replaced with three different versions of an expressioncassette containing the Rous sarcoma virus-long terminal repeat(RSV-LTR) promoter driving expression of a bicistronic mRNA containingat the 5′ end the Ad2 E3 19 kDa anti-immunity gene product and at the 3′end the Ad5 E3 14.7 kDa anti-inflammatory gene product. One additionalvirus was constructed by deleting the 19 kDa cDNA fragment by Bst BI(NEB) fragment deletion. This virus, designated Ad_(GV)CFTR.11(D),contains the RSV-LTR promoter driving expression of a monocistronic mRNAcontaining only the E3 14.7 kDa anti-inflammatory gene product.

The Spe I (27082)—Nde I (31089) fragment from pGBSΔE3 (Example 5) wassubcloned into pUC 19 by first cloning the Eco RI (27331)—Nde I (31089)fragment into identical sites in the pUC 19 polylinker. A Hin dIII(26328)—Eco RI (27331) fragment generated from pGBS was then cloned intothe Eco RI site of this clone to generate pHNΔE3. Using appropriateprimers, a PCR fragment with flanking Xba I sites was generatedcontaining the RSV-LTR promoter, the Ad2 E3 19 kDa gene product, and theAd5 E3 14.7 kDa gene product. The amplified fragment was digested withXba I and subcloned into pUC 19 to generate pXA. After analysis of theXba I fragment, the fragment was ligated into pHNΔE3 to generate pHNRA.

Using appropriate primers, two PCR fragments with flanking Bst BI siteswere generated that encode internal ribosomal entry sites (IRES), whichare known to enhance the translation of mRNAs that contain them (Joblinget al., Nature, 325, 622-625 (1987); Jang et al., Genes and Development,4, 1560-1572 (1990)). One fragment (version B) contains a 34 bp IRESfrom the untranslated leader of the coat protein mRNA of alfalfa mosaicvirus (AMV RNA 4 leader) (Jobling et al., supra). The other fragment(version C) contains a 570 bp IRES from the 5′ nontranslated region ofencephalomyocarditis virus (EMCV) mRNA (Jang et al., supra). Each Bst BIfragment from version B or C was cloned in place of the Bst BI fragmentin pXA. The resulting plasmids, named pXB and pXC, respectively, weremoved into pHNΔE3 to generate PHNRB and pHNRC, respectively, aftersequence analysis of the Xba I fragments.

The Spe I (27082)—Nde I (31089) fragment from pGBSΔE30RF6 was replacedwith the Spe I—Nde I fragments from pHNRA, pHNRB, pHNRC and pHNRD togenerate pGV11A, pGV11B, pGV11C and pGV11D, respectively.

The PGBV plasmid DNA was linearized with Bam HI and Sal I and mixed withthe purified left arm DNA fragment in varying concentrations to giveabout 20 μg total DNA, using salmon sperm or calf thymus DNA (LifeTechnologies, Gaithersburg, Mass.) to bring the amount of DNA to about20 μg as needed. The mixed fragments were then transfected into 293cells using standard calcium phosphate techniques (Graham et al.,supra).

Five days after transfection, the cell monolayer was harvested byfreeze-thawing three times. The resulting hybrid virus was titered onto293 cells and isolated plaques were picked. The process of plaqueisolation was repeated twice more to ensure a single recombinant virusexisted in the initial plaque stock. The plaque isolate stock was thenamplified to a large viral stock according to standard virologytechniques as described in Burlseson et al., Virology: a LaboratoryManual, Academic Press Inc. (1992).

FIG. 2 is a set of schematic diagrams of the various AD_(GV)CFTR.11viral vectors. The diagrams are aligned with that of AD_(GV)CFTR.10L forcomparison.

EXAMPLE 3

This example describes the generation of Ad_(GV)CFTR.12.

Ad_(GV).12 is characterized by complete elimination of the E4 region.This large deletion allows for insertion of up to about 10 kb ofexogenous DNA. More importantly, another region of the genome has becomeaccessible for introduction of foreign gene expression cassettes. Thisdeletion now enables the incorporation of larger expression cassettesfor other products. For example, soluble receptors, i.e., TNF or IL-6without a transmembrane domain so that they are now not attached to themembrane, and antisense molecules, e.g., those directed against cellcycle regulating products, such as cdc2, cdk kinases, cyclins, i.e.,cyclin E or cyclin D, and transcription factors, i.e., E2F or c-myc, toeliminate inflammation and immune responses.

pGV11(O) is altered to produce a right arm plasmid in which the entireE4 region is deleted. The resulting plasmid in which the entire E3 andE4 regions are deleted is named pGV12(O). This is done by introducing aPac I restriction site at the Afl III site at 32811 and the Bsa I siteat 35640. Deletion of the Pac I fragment between these two siteseffectively eliminates all of the E4 sequences including the E4 TATAelement within the E4 promoter and the E4 poly A site.

Virus construction is performed as previously described except that the293/E4 cell line or the 293/ORF6 cell line is used. The left arm fromAD_(GV)CFTR.10L, the right arm pGV12(O) plasmid, and all other generaltechniques are as described in Example 2. Since E4 contains essentialgene products necessary for viral growth, the resulting E4 deletionmutant virus cannot grow in the absence of exogenously expressed E4.Therefore, all manipulations for viral construction are carried out inthe new 293/E4 cell line or 293/ORF6 cell line (described in Examples 8and 9, respectively). The resulting virus is Ad_(GV)CFTR.12.

EXAMPLE 4

This example describes the generation of Ad_(GV)CFTR.13.

Ad_(GV).13 is characterized by not only complete elimination of E1, andE4 (as in AD_(GV).12) but also complete elimination of E2A. The completecoding region of E2A is deleted by fusing together the DNA from two E2Amutant viruses, namely H5in800 and H5in804, containing insertions of ClaI restriction sites at both ends of the open reading frame (Vos et al.,Virology, 172, 634-642 (1989); Brough et al., Virology, 190, 624-634(1992)). The Cla I site of H5in800 is between codons 2 and 3 of thegene, and the Cla I site of H5in804 is within the stop codon of the E2Agene. The resultant virus contains an open reading frame consisting of23 amino acids that have no similarity to the E2A reading frame. Moreimportantly, this cassette offers yet another region of the virus genomeinto which a unique gene can be introduced. This can be done byinserting the gene of interest into the proper reading frame of theexisting mini-ORF or by introducing yet another expression cassettecontaining its own promoter sequences, polyadenylation signals, and stopsequences in addition to the gene of interest.

Adenovirus DNA is prepared from H5in800 and H5in804. After digestionwith the restriction enzyme Hin dIII (NEB), the Hin dIII A fragmentsfrom both H5in800 and H5in804 are cloned into pKS+ (Stratagene). Theresulting plasmids are named pKS+H5in800Hin dIIIA and pKS+H5in804HindIIIA, respectively. The Cla I (NEB) fragment from pKS+H5in800Hin dIIIAis then isolated and cloned in place of the identical Cla I fragmentfrom PKS+H5in804Hin dIIIA. This chimeric plasmid, pHin dIIIAΔE2Aeffectively removes all of the E2A reading frame as described above. Atthis point, the E2A deletion is moved at Bam HI (NEB) and Spe I (NEB)restriction sites to replace the wild-type sequences in pGV12(O) toconstruct pGV13(O).

Ad_(GV)CFTR.13 virus is constructed as previously described by usingAd_(GV)CFTR.10 left arm DNA and pGV13(O) right arm plasmid DNA. However,the recipient cell line for this virus construction is the triplecomplementing cell line 293/E4/E2A.

EXAMPLE 5

This example describes the generation of pGBSΔE3.

This plasmid was generated to remove the majority of the E3 regionwithin PGBS, including the E3 promoter and existing E3 genes, to makeroom for other constructs and to facilitate introduction of E3expression cassettes. This plasmid contains a deletion from 28331 to30469.

A PCR fragment was generated with Ad5s(27324) and A5a(28330)X as primersand pGBS as template. The resulting fragment was digested with Eco RI(27331) and Xba I (28330) and gel-purified. This fragment was thenintroduced into pGBS at the Eco RI (27331) and Xba I (30470) sites.

EXAMPLE 6

This example describes the generation of pGBSΔE3ΔE4.

A large deletion of the Ad5 E4 region was introduced into pGBSΔE3 tofacilitate moving additional exogenous sequences into the adenoviralgenome. The 32830-35566 E4 coding sequence was deleted.

A Pac I site was generated in place of the Mun I site at 32830 bytreating pGBS Mun I-digested DNA with Klenow to blunt-end the fragmentand by ligating a Pac I linker to this. The modified DNA was thendigested with Nde I and the resulting 1736 bp fragment (Nde I 31089—PacI 32830) was gel-purified. A PCR fragment was prepared using A5 (35564)P(IDT, Coralville, Iowa) and T7 primers (IDT, Coralville, Iowa) and pGBSas template. The resulting fragment was digested with Pac I and Sal I togenerate Pac I 35566—Sal I 35935. A Sma I site within the polylinkerregion of pUC 19 was modified to a Pac I site by ligating in a Pac Ilinker. The Pac I 35566—Sal I 35935 fragment was moved into the modifiedpUC 19 vector at Pac I and Sal I sites, respectively, in the polylinkerregion. The modified Nde I 31089—Pac I 32830 fragment was moved into thepUC 19 plasmid, into which the Pac I 35566—Sal I 35935 fragment alreadyhad been inserted, at Nde I and Pac I sites, respectively. The Nde I31089—Sal I 35935 fragment from the pUC 19 plasmid was purified by gelpurification and cloned in place of the respective Nde I and Sal I sitesin pGBSΔE3 to yield pGBSΔE3ΔE4.

EXAMPLE 7

This example describes the generation of pGBSΔE3ORF6.

The Ad5 894 bp E4 ORF-6 gene was placed 3′ of the E4 promoter inpGBSΔE3ΔE4. ORF-6 is the only absolutely essential E4 product necessaryfor virus growth in a non-E4 complementing cell line. Therefore, thisproduct was re-introduced into the right arm plasmid (Example 2) underits own promoter control so that Ad_(GV)CFTR.11 virus can be propagatedin 293 cells.

A PCR fragment was generated using A5s(33190)P (32 bp;5′CACTTAATTAAACGCCTACATGGGGGTAGAGT3′) (SEQ ID NO:1) and A5a(34084)P (34bp; 5′CACTTAATTAAGGAAATATGACTACGTCCGGCGT3′) (SEQ ID NO:2) as primers(IDT, Coralville, Iowa) and PGBS as template. This fragment was digestedwith Pac I and gel-purified. The product was introduced into the singlePac I site in pGBSΔE3ΔE4 to generate pGV11(O), which was the plasmidthat was E3-modified for expression of the 19 kDa and 14.7 kDa Ad E3products.

EXAMPLE 8

This example describes the generation of the 293/E4 cell line.

The vector pSMT/E4 was generated as follows. A 2752 bp Mun I (site 32825of Ad2)—Sph I (polylinker) fragment was isolated from pE4(89-99), whichis a pUC19 plasmid into which was subcloned region 32264-35577 from Ad2,blunt-ended with Klenow, and treated with phosphatase (NEB). The 2752 bpMun I—Sph I fragment was then ligated into pMT010/A⁺ (McNeall et al.,Gene, 76, 81-89 (1989)), which had been linearized with Bam HI,blunt-ended with Klenow and treated with phosphatase, to generate theexpression cassette plasmid, pSMT/E4.

The cell line 293 (ATCC CRL 1573; American Type Culture Collection,Rockville, Md.) was cultured in 10% fetal bovine serum Dulbecco'smodified Eagle's medium (Life Technologies, Gaithersburg, Mass.). The293 cells were then transfected with pSMT/E4 linearized with Eco RI bythe calcium phosphate method (Sambrook et al., Molecular Cloning: aLaboratory Manual, Cold Spring Harbor Laboratory Press (1989)).Approximately 24-48 hours post-transfection, medium (as above)containing 100 μM methotrexate and amethopterin (Sigma Chemical Co., St.Louis, Mo.) was added. The presence of methotrexate in the mediumselects for expression of the dihydrofolate reductase (DHFR) gene, whichis the selectable marker on the pSMT/E4 plasmid.

The normal cell DHFR gene is inhibited by a given concentration ofmethotrexate (cell type-specific), causing cell death. The expression ofthe additional DHFR gene in transfected cells containing pSMT/E4provides resistance to methotrexate. Therefore, transfected cellscontaining the new genes are the only ones that grow under theseconditions (for review, see Sambrook et al., supra).

When small colonies of cells formed from the initial single cell havingthe selectable marker, they were clonally isolated and propagated (forreview, see Sambrook et al., supra). These clones were expanded toproduce cell lines that were screened for expression of the product—inthis case, E4—and screened for functionality in complementing defectiveviruses—in this case, both E1 and E4 defective viruses.

The result of this process produced the first 293/E4 cell lines capableof complementing adenoviral vectors defective in both E1 and E4functions, such as Ad_(GV)CFTR.12.

EXAMPLE 9

This example describes the generation of the 293/ORF-6 cell line.

The primers A5s(33190)P and A5a(34084)P were used in a polymerase chainreaction (PCR) (PCR Protocols, A guide to Methods and Aoplications,Innis et al., eds., Academic Press, Inc. (1990)) to amplify the ORF-6gene of Ad5 E4 and generate. Pac I sites at the ends for cloning. Theamplified fragment was blunt-ended with Klenow and cloned intopCR-Script SK(+) (Stratagene, La Jolla, Calif.). The resulting plasmid,pCR/ORF-6, was sequenced and then the ORF-6 insert was transferred intothe pSMT/puro expression vector, which was generated by ligation of ablunt-ended Eco RI—Hin dIII fragment containing the SMT promoter intothe blunt-ended Mlu I-Hin dIII site in pRCpuro, to generate pSMT/ORF-6.

The 293 cell line was cultured and transfected with pSMT/ORF-6 asdescribed in Example 8, except that the transfected cells were placedunder selection for the puromycin resistance gene, which allows cellsthat express it to grow in the presence of puromycin. Colonies oftransformed cells were subcloned and propagated and were screened asdescribed in Example 8.

This cell line is suitable for complementing vectors that are deficientin the E1 and E4 region, such as the Ad_(GV)CFTR.12 series of vectors.

EXAMPLE 10

This example describes the generation of the 293/E4/E2A cell line. The293/E4/E2A cell line allows E1, E4 and E2A defective viral vectors togrow.

The E2A expression cassette for introduction into 293/E4 cells isproduced as follows. The first step is to alter surrounding bases of theATG of E2A to make a perfect Kozak consensus (Kozak, J. Molec. Biol.,196, 947-950 (1987)) to optimize expression of E2A. Two primers aredesigned to alter the 5′ region of the E2A gene. Ad5s(23884), an 18 bpoligonucleotide (5′gCCgCCTCATCCgCTTTT3′) (SEQ ID NO:3), is designed toprime the internal region flanking the Sma I site of the E2A gene.DBP(ATG)R1, a 32 bp oligonucleotide(5′CCggAATTCCACCATggcgAgtcgggAAgAgg3′) (SEQ ID NO:4), is designed tointroduce the translational consensus sequence around-the ATG of the E2Agene modifying it into a perfect Kozak extended consensus sequence andto introduce an Eco RI site just 5′ to facilitate cloning. The resultingPCR product using the above primers is digested with Eco RI and Sma I(NEB) and cloned into the identical polylinker sites of pBluescriptIIKS+ (Stratgene, La Jolla, Calif.). The resulting plasmid is namedpKS/ESDBP.

A Sma I-Xba I fragment is isolated from pHRKauffman (Morin et al., Mol.Cell. Biol., 9, 4372-4380 (1989)) and cloned into the corresponding SmaI and Xba I sites of pKS/ESDBP to complete the E2A reading frame. Theresulting plasmid is named pKSDBP. In order to eliminate all homologoussequences from vector contained within the expression cassette, the KpnI to Dra I fragment from pKSDBP is moved into corresponding Kpn I andPme I sites in PNEB193 (NEB) in which the Eco RI sites in the polylinkerhave been destroyed (GenVec). The resulting clone, pE2A, contains all ofthe E2A reading frame without any extra sequences homologous to the E2Adeleted vector in Example 4.

A 5′ splice cassette is then moved into pE2A to allow proper nuclearprocessing of the mRNA and to further enhance expression of E2A. To dothis, pRK5, described in Example 1, is digested with Sac II (NEB),blunt-ended with Mung Bean nuclease (NEB), and digested with Eco RI(NEB). The resulting approx. 240 bp fragment of interest containing thesplicing signals is cloned into the Cla I (blunt-ended with Klenow) toEco RI sites of pE2A to generate p5′E2A. The blunt-ended (Klenow) Sal Ito Hin dIII fragment from p5′E2A containing the E2A sequences is movedinto the blunt-ended (Klenow) Xba I site of pSMT/puro and pSMT/neo. Theresulting E2A is named pKSE2A.

The Xba I fragment from pKSE2A that contained all the E2A gene is movedinto the Xba I site of pSMT/puro and pSMT/neo. The resulting E2Aexpression plasmids, pSMT/E2A/puro and pSMT/E2A/neo, are transfectedinto 293/E4 and 203/ORF-6 cells, respectively. Cells transfected withpSMT/E2A/puro are selected for growth in standard media plus puromycinand cells transfected with pSMT/E2A/neo are selected for growth instandard media plus G418. Clonal expansion of isolated colonies is asdescribed in Example 8. The resulting cell lines are screened for theirability to complement E1, E4 and E2A defective viral vectors.

These cell lines are suitable for complementing vectors that aredeficient in the E1, E4 and E2A regions of the virus, such as thosedescribed in the Ad_(GV)CFTR.13 series of viral vectors.

EXAMPLE 11

This example describes the generation of complementing cell lines usingthe cell line A549 (ATCC) as the parental line.

Ad2 virus DNA is prepared by techniques previously described. Thegenomic DNA is digested with Ssp I and Xho I and the 5438 bp fragment ispurified and cloned into Eco RV/Xho I sites of pKS+ (Stratagene) toproduce pKS341-5778. After diagnostic determination of the clone, an XhoI (blunt-ended with Klenow) to Eco RI fragment is moved into Nru I(blunt) to Eco RI sites in pRC/CMVneo to produce pE1neo. Transformationof A549 cells with this clone yields a complementing cell line (similarto 293), wherein additional expression cassettes can be introduced, in amanner similar to that described for the 293 cell, to producemulticomplementing cell lines with excellent plaqueing potential.

All references, including publications and patents, cited herein arehereby incorporated by reference to the same extent as if each referencewere individually and specifically indicated to be incorporated byreference and were set forth in its entirety herein.

While this invention has been described with emphasis upon preferredembodiments, it will be obvious to those of ordinary skill in the artthat the preferred embodiments may be varied. It is intended that theinvention may be practiced otherwise than as specifically describedherein. Accordingly, this invention includes all modificationsencompassed within the spirit and scope of the appended claims.

4 32 base pairs nucleic acid single linear DNA (synthetic) 1 CACTTAATTAAACGCCTACA TGGGGGTAGA GT 32 34 base pairs nucleic acid single linear DNA(synthetic) 2 CACTTAATTA AGGAAATATG ACTACGTCCG GCGT 34 18 base pairsnucleic acid single linear DNA (synthetic) 3 GCCGCCTCAT CCGCTTTT 18 32base pairs nucleic acid single linear DNA (synthetic) 4 CCGGAATTCCACCATGGCGA GTCGGGAAGA GG 32

What is claimed is:
 1. A system comprising: (i) an adenoviral vectorcomprising an adenoviral genome having a deficiency in one or moreessential gene functions of the E1 region of the adenoviral genome and adeficiency in one or more essential gene functions in either or both ofthe E2A region and the E4 region of the adenoviral genome, and (ii) acell having a cellular genome that complements in trans for thedeficiency in one or more essential gene functions of the E1 region ofthe adenoviral genome and the deficiency in one or more essential genefunctions in either or both of the E2A region and the E4 region of theadenoviral genome, wherein there is no overlap between the cellulargenome and the adenoviral genome to mediate a recombination eventbetween the cellular genome and the adenoviral genome.
 2. The system ofclaim 1, wherein the adenoviral vector comprises an adenoviral genomehaving a deficiency in one or more essential gene functions of theE1region of the adenoviral genome and a deficiency in one or moreessential gene functions of the E4 region of the adenoviral genome andthe cell has a cellular genome that complements in trans for thedeficiency in one or more essential gene functions of the E1 region ofthe adenoviral genome and the deficiency in one or more essential genefunctions of the E4 region of the adenoviral genome.
 3. The system ofclaim 2, wherein the cellular genome comprises at least open readingframe (ORF) 6 of the E4 region of the adenoviral genome.
 4. The systemof claim 3, wherein the cellular genome comprises at least ORF6 and noother ORF of the E4 region of the adenoviral genome.
 5. The system ofclaim 4, wherein the cellular genome comprises an E1A coding sequenceand an E1B coding sequence.
 6. A method of propagating an adenoviralvector, which method comprises (a) providing an adenoviral vectorcomprising an adenoviral genome having a deficiency in one or moreessential gene functions of the E1 region of the adenoviral genome and adeficiency in one or more essential gene functions in either or both ofthe E2A region and the E4 region of the adenoviral genome, (b) providinga cell comprising a cellular genome that complements in trans for thedeficiency in one or more essential gene functions of the E1 region ofthe adenoviral genome and the deficiency in one or more essential genefunctions in either or both of the E2A region and the E4 region of theadenoviral genome, wherein there is no overlap between the cellulargenome and the adenoviral genome to mediate a recombination eventbetween the cellular genome and the adenoviral genome, and (c)propagating the adenoviral vector in the cell.
 7. The method of claim 6,wherein the adenoviral vector comprises an adenoviral genome having adeficiency in one or more essential gene functions of the E1 region ofthe adenoviral genome and a deficiency in one or more essential genefunctions of the E4 region of the adenoviral genome, and the cell has acellular genome that complements in trans for the deficiency in one ormore essential gene functions of the E1 region of the adenoviral genomeand the deficiency in one or more essential gene functions in the E4region of the adenoviral genome.
 8. The method of claim 7, wherein thecellular genome comprises at least open reading frame (ORF) 6 of the E4region of the adenoviral genome.
 9. The method of claim 8, wherein thecellular genome comprises at least ORF6 and no other ORF of the E4region of the adenoviral genome.
 10. The method of claim 9, wherein thecellular genome comprises an E1A coding sequence and an E1B codingsequence.